Ethanol production by high performance bacterial fermentation

ABSTRACT

The present invention is directed to the preparation of ethanol by bacterial fermentation. It makes use of a microorganism capable of producing ethanol and the process is carried out in two stages. In the first stage a bacterial cell suspension is produced together with ethanol in an ethanol concentration range that does not substantially inhibit production of the bacterial cells in a medium containing a source of nitrogen and a source of carbon. Ethanol is then produced in the absence of substantial bacterial cell production by the addition of fermentable sugar to the bacterial cell suspension which is produced in the first stage. The preferred microorganism is a member of the genus Zymomonas.

This application is a continuation of application Ser. No. 641,364,filed Aug. 15, 1984, now U.S. Pat. No. 4,647,534, issued Mar. 3, 1987,which is a continuation of application Ser. No. 217,066, filed Sept. 16,1980, now abandoned, which in turn is a continuation-in-part ofapplication Ser. No. 184,508, filed Sept. 5, 1985, now abandoned.

The present invention relates to a process for the production of ethanolby fermentation. In particular it relates to the production of ethanolby a process of bacterial fermentation, in which there is improvedfermentation efficiency and product yield as compared to prior artmethods.

The traditional process of fermentation is carried out in a conventionalbatch operation utilizing yeast as the fermenting organism. To increasethe efficiency a variation of the batch operation occasionally includesrecycling of the yeast cells by systems such as sedimentation,centrifugation, or ultrafiltration. Normally this batch operation isconducted in two stages. The first stage involves propagation of theyeast and is referred to as the growth stage. The second stage involvesthe anaerobic process of ethanol production which is accompanied by adepletion of the oxygen. Further propagation of the yeast occurs duringthe anaerobic process of ethanol production.

Typically, a yeast inoculum is prepared in stage one. The requirementsfor maximum yeast reproduction are adequate amounts of carbon, nitrogen,minerals and oxygen, a pH in the range of 3.5 to 4.5, and a temperaturein the range of 29°-35° C. Aerobic growth conditions define a system formore efficient production of yeast but under which no ethanol isreproduced.

Stage two is the fermentation stage where the alcohol is actuallyproduced by the yeast from the fermentable sugars. The yeast inoculumproduced in stage (i) is used to seed a large fermenter previouslyfilled with the substrate, which may be molasses, corn, etc., adjustedto the appropriate pH, temperature and sugar concentration. Theinoculation rate can be 5 to 10 million cells per ml and during thefermentation the viable cell count can increase to 150-200 million cellsper ml. Heat produced is controlled through the use of cooling coils. Atthese yeast levels, a final ethanol concentration of about 9 to 11%(v/v) can be obtained in 30 to 70 hours with batch fermentation.Increasing the yeast content, as is the case with cell recycle, canconsiderably reduce the time required for completion of thefermentation. For example, with a cell density of 800 to 1000 millioncells per ml, it is possible to reduce the fermentation time to 4 to 10hours.

It is recognized that, although conditions of strict anaerobiosispromote maximum production of ethanol, growth of yeast is suppressed.Inherent in this fermentation process is the coupling of growth with therate of alcohol production. Consequently, in order to optimize ethanolproduction, either the degree of aeration must be finely controlled, orthe medium must be supplied with growth-promoting supplements (forexample, ergosterol or unsaturated fatty acids such as oleic acid).

Accordingly, the present invention describes a process for theproduction of ethanol by bacterial further that yeast fermentation. Theprocess utilizes an organism which is capable of producing ethanol suchas the bacterium Zymomonas as the fermenting organism. A number ofdifferent strains of Zymomonas mobilis may be used; for example ATCC29191, ATCC 10988, etc. The process consists of two stages, an initialstage for the production of biomass, and a second stage for theproduction of ethanol. Unlike the prior art methods, the growth stage inthis process occurs anaerobically and is accompanied by the productionof ethanol. The fermentation stage of the Z. mobilis process resemblesthat of the prior art method in the respect that both occur underanaerobic conditions. However, in the bacterial process according to thepresent invention the production of ethanol is essentially uncoupledfrom growth (i.e. production of biomass). Thus, during the production ofethanol from such a "resting culture", only a small proportion of thesubstrate is converted to biomass and ethanol production is maximized.It follows that production of ethanol in the absence of growth with thebacterial process can be achieved simply by the addition of fermentablesugar and this system thereby is capable of yielding higher alcohollevels than the prior art process. In the prior art process high alcohollevels prevent growth and therefore alcohol production since this occursonly to a limited extent without growth.

Accordingly, the present invention comprises a process for producingethanol by fermentation, which comprises culturing a microorganismcapable of producing ethanol in two stages comprising

(i) producing a bacterial cell suspension together with ethanol in anethanol concentration range that does not substantially inhibitproduction of the microbial cells in a medium containing a source ofnitrogen and a source of carbon and

(ii) producing ethanol in the absence of bacterial cell production bythe addition of fermentable sugar to the bacterial cell suspensionproduced in (i).

STAGE (i)

Growth of Zymomonas mobilis is conducted in an aqueous nutrient medium.The medium may be either a natural nutrient medium, a synthetic culturemedium or a semi-synthetic culture medium as long as a suitablecarbohydrate source and the essential nutrients for the growth of themicroorganism are present. Although the growth stage in the process isnormally used to generate sufficient quantities of biomass for theoperation of the fermentation stage, the biomass may also be recycledwith concomitant reduction in the essential nutrients required by theprocess.

In general, conditions which may be used to promote growth of Z. mobilisare: adequate carbon supply, adequate nitrogen supply, appropriateorganic growth factors, adequate mineral supply, essentially anaerobicconditions, agitation of the culture, temperature in the range of20°-40° C., and pH in the range of 4 to 8.

As carbon source for both the growth and the fermentation stages of theprocess various carbohydrates may be used. The carbohydrates include,for example, sugars such as glucose, fructose, sucrose; molasses; starchhydrolysate; cellulose hydrolysate; etc. These substances may be usedeither singly or in mixtures of two or more. As a nitrogen source,various kinds of inorganic or organic salts or compounds, for exampleammonium salts such as ammonium chloride, ammonium sulfate, etc., ornatural substances containing nitrogen, such as yeast extract, caseinhydrolysate, corn steep liquor etc., or amino acids such as glutamicacid may be employed. These substances may also be used either singly orin combinations of two or more.

Inorganic compounds which may be added to the culture medium includemagnesium sulfate, potassium monohydrogen phosphate, potassiumdihydrogen phosphate, sodium chloride, magnesium sulfate, calciumchloride, iron chloride, magnesium chloride, zinc sulfate, cobaltchloride, copper chloride, borates, molybdates, etc.

Organic compounds which may be desirable for the operation of theprocess include, for example, vitamins such as biotin, calciumpantothenate, and the like, or organic acids such as citric acid oramino acids such as glutamic acid.

The microorganisms may be grown under the commonly-named operatingconditions of either batch or continuous culture, with or without cellrecycle in either case. The culturing or fermentation is conducted underessentially anaerobic conditions with agitation of a submerged culture,at a temperature of for example 20°-40° C., and a pH of for example 4.0to 8.0. The preferred conditions are a temperature of about 30° C. and apH of about 5.5. It may be desirable to add certain pH regulating agentsto the medium during the course of culture fermentation, such as sodiumhydroxide, hydrochloric acid, or the like.

STAGE (ii)

In the second stage of the process a broth of high ethanol content isproduced by fermentation. Additional carbon in amounts such that thefinal ethanol concentration reaches the desired level is added to thespent broth containing the bacterial biomass. The carbon may be addedeither stepwise or continuously as a concentrated solution, but mustnever exceed approximately 6% (w/v). The options available as possiblecarbon sources for fermentation are described in stage (i) of theprocess.

The fermentation may be operated under the process conditions ofso-called fed-batch or continuously, with or without cell recycle ineither case. Process parameters for good production of ethanol includetemperature in the range of 20°-40° C., pH in the range of 4 to 8,mixing of the fermenting broth, and essentially anaerobic conditions.The preferred temperature is about 28° to 33° C., and the preferred pHis about 5.5. It may be necessary or desirable to add pH regulatingagents as described in stage (i). Anaerobic conditions may be maintainedby bubbling a slow stream of nitrogen through the broth. Occassionallyadditional nutrients as described in stage (i) may be added to thefermenter.

The invention will be better understood by reference to the followingexamples which illustrate the invention.

EXAMPLE 1

1200 ml of a fermentation medium having the following compositions wereplaced in a 2 liter fermenter vessel:

Glucose: 10% (w/v)

Yeast extract (Difco): 1.5%

KH₂ PO₄ : 0.375%

NH₄ Cl: 0.24%

MgSO₄ : 0.15%

Citric Acid: 1.5 mM

CaCl₂.2H₂ O: 150 μM

FeCl₃.6H₂ O: 135 μM

MnCl₂.4H₂ O: 75 μM

ZnSO₄.7H₂ O: 38 μM

CoCl₂.6H₂ O: 15 μM

CuCl₂.2H₂ O: 8 μM

H₃ BO₃ : 8 μM

MoO₃ : 15 μM

Biotin: 1.5 mg/L

Ca Pantothenate: 2.25 mg/L

10 ml of seed culture of Zymomonas mobilis ATCC 29191 grown in a mediumof the above composition was added to the above fermentation medium.Cultivation was carried out at a temperature of 30° C. with a nitrogenflow of 0.5 SCFH into the culture and with stirring at a rate of 300rpm. The pH was maintained at 5.5 with 2N KOH.

After 15 hours growth had terminated with the concentration of bacterialcells at 4 g/L (dry weight) and the ethanol concentration at 4.65%(w/v).

After the termination of growth, the second stage of the process wasbegun by pumping an additional 340 g of glucose dissolved in 600 ml ofwater into the fermenter vessel over a period of 8.5 hours. Completeutilization of the sugar had occurred by 9.5 hours. The ethanolconcentration at the end of the second stage was 11.6%.

EXAMPLE 2

12 liters of a fermentation medium having the following composition wereplaced in a 14 liter fermenter vessel:

Glucose: 10% (w/v)

Yeast extract (Difco): 1.0%

KH₂ PO₄ : 0.25%

NH₄ Cl: 0.16%

MgSO₄ : 0.1%

Citric Acid: 1.0 mM

CaCl₂.2H₂ O: 100 μM

FeCl₃.6H₂ O: 90 μM

MnCl₂.4H₂ O: 50 μM

ZnSO₄.7H₂ O: 25 μM

CoCl₂.6H₂ O: 10 μM

CuCl₂.2H₂ O: 5 μM

H₃ BO₃ : 5 μM

MoO₃ : 10 μM

Biotin: 1.0 mg/L

Ca Pantothenate: 1.5 mg/L

50 ml of seed culture of Zymomonas mobilis ATCC 29191 grown in a mediumof the above composition was added to the above fermentation medium.Cultivation was carried out at a temperature of 30° C. with a nitrogenflow into the fermenter and with stirring at a rate of 300 rpm. The pHwas maintained at 5.5 with 8N KOH.

After growth had terminated, the bacterial cells were collected bycentrifugation and resuspended in fresh medium of the above compositionto give a bacterial cell concentration of 20 g/L.

1200 ml of the concentrated bacterial cell suspension were placed in a 2liter fermenter vessel. Stage (ii) of the process was initiated by theaddition of 32 ml of a 75% glucose solution to the fermenter. A further450 ml of 75% glucose was then pumped into the fermenter vessel over thecourse of 2 hours. Utilization of the sugar was complete after 2.5 hoursgiving an ethanol concentration of 10.1%.

Stage (ii) of the process was repeated a further four times using thebacterial cells generated by a single stage (i) of the process. Aftereach passage through stage (ii), the bacterial cells were collected bycentrifugation and resuspended in 1200 ml of fresh medium of the abovecomposition. Glucose was added in the manner described above for thefirst passage of the cells through state (ii). In each of the fourcycles utilizing the same biomass, the glucose was completely utilizedafter 2.5 hours with an ethanol concentration of approximately 10% beingobtained in each instance.

EXAMPLE 3

Stage (i) of the process was conducted in a 740 ml fermenter containing320 ml of the fermentation medium described in Example 2 and stage (ii)was conducted in a 2 liter fermenter containing 1740 ml of the samemedium.

The stage (i) fermenter was inoculated with 7 ml of seed culture ofZymomonas mobilis ATCC 29191 grown in a medium of the compositiondescribed in example 2; stage (ii) was inoculated with 12 ml of the sameseed culture. Cultivation was carried out at 30° C. with a nitrogen flowinto the fermenters and with a stirring rate of 300 rpm. The pH of eachfermenter was maintained at 5.5 with 2N KOH.

After growth had terminated two pumps were employed to make the systemcontinuous. The first pump supplied sterile medium containing 12.6%glucose (other components as in Example 2) at a flow rate of 78 ml/hr.Under these conditions, the bacterial cell concentration in the stage(i) fermenter was 4.5 g/L, the ethanol concentration was 5.3% and theresidual glucose concentration was 0.6%. The volume of the stage (i)fermenter was maintained at 320 ml by continuously transferring cultureto the stage (ii) fermenter at a flow rate of 78 ml/hr. The second pumpsupplied a sterile 70% glucose solution to the stage (ii) fermenter at aflow rate of 24 ml/hr. giving a total flow in and out of the fermenterof 102 ml/hr. Under these conditions, the bacterial cell concentrationin the stage (ii) fermenter was 4.4 g/L, the ethanol concentration was9.9% and the residual glucose concentration was 1.2%.

EXAMPLE 4

In this example Zymomonas mobilis ATCC 29191 was cultured in twofermenters which were operated in tandem in a similar manner asdescribed in Example 3. In this example, however, the biomass containedin the effluent from the second fermenter was retained and added back tothe second fermenter. Biomass recycle to the second fermenter wasachieved by continuously processing the contents through a PelliconUltrafilter Cassette® (Millipore Corp.) containing 6 sq. ft. of HA typeMillipore filters. The culture medium used in this example was of thesame composition as examples 2 and 3 except the yeast extract was 0.5%(w/v). The constant volume of the first fermenter (stage (i)) was 1600ml. It was fed culture medium containing glucose (131 g/L) at a constantrate of 414 ml/hr. The temperature and pH were maintained at 30° C. and5.5 respectively. The steady-state concentration of biomass in the firstfermenter was 4.2 g D.W/L and the ethanol was 5.1% (w/v).

The effluent from the first fermenter was fed continuously and directlyto a second fermenter which was operated at a constant volume of 760 ml.A solution of 60% w/v glucose was pumped into the second fermenter at aconstant rate of 120 ml/hr. The effluent from the second fermenter wasprocessed through the filtration system with the cells being returned tothe second fermenter. The biomass level in the second fermenter was 48 gD.W/L and was kept relatively constant by removing biomass at a rate ofabout 27 ml/hr. The concentration of ethanol in the cell-free filtrateleaving the system was 10.4% w/v and the residual sugar was 1.7%.Operation of the two stage system at an elevated biomass level in thesecond fermenter as a consequence of effluent filtration and biomassrecycling, resulted in an increased ethanol productivity of the secondfermenter (product produced per unit volume per unit time).

I claim:
 1. A continuous process for producing ethanol by fermentationin an aqueous medium which comprises cultivating Zymomonas mobilis ATCC29191 in two stages in order to increase ethanol concentration in thefermentation medium, as compared to ethanol concentration in afermentation medium obtained by continuous cultivation of said Zymomonasmobilis microorganism in a single stage, said process consistingessentially of(i) producing a bacterial biomass comprising a cellsuspension of said Zymomonas together with ethanol in an ethanolconcentration range which does not substantially inhibit production ofsaid Zymomonas cells in a medium containing glucose and a source ofnitrogen; and (ii) producing ethanol in the absence of substantialproduction of said Zymomonas cells, by the addition of a mediumcontaining glucose to the bacterial cell suspension produced in (i),wherein sugar concentration in stage (ii) does not exceed about 6% w/v;wherein in step (i) the source of nitrogen is a mineral salt mediumcontaining NH₄ + ions as the sole source or a mineral salt mediumcontaining NH₄ + ions supplemented with an organic source of nitrogen,and ethanol reaches a steady state concentration of about 10% w/v in thebiomass in (ii).
 2. A process as claimed in claim 1 wherein the twostages of the process are conducted in two fermenters with the bacterialcells produced by a continuous culture in the first fermenter beingcontinuously transferred to a second fermenter.
 3. A process as claimedin claim 2 wherein stage (ii) of the process is conducted in a fermenterin which the contents have a substantially constant volume, saidfermenter having a continuous feed of the medium required for the secondstage of the process.
 4. A process as claimed in claim 3 wherein ethanolis recovered from biomass and stage (ii) is repeated up to four times.5. A process as claimed in claim 2 wherein a portion of the bacterialcells in the second fermenter is removed therefrom and separated fromthe fermentation broth by continuous filtration or sedimentation andreturned to stage (ii) to thereby increase ethanol productivity in stage(ii).
 6. A process as claimed in claim 5 wherein stage (ii) is carriedout at a temperature of about 28° to 33° C. and at a pH of about 5.5. 7.A process as claimed in claim 6 wherein stage (i) is carried out at atemperature of about 30° C. and a pH of about 5.5.
 8. A process asclaimed in claim 6 wherein the fermenter in stage (i) and the fermenterin stage (ii) have a working volumetric ratio of about 2:1.
 9. A processas claimed in claim 6 wherein ethanol is recovered from biomass andstage (ii) is repeated up to four times.
 10. A process as claimed inclaim 1 wherein ethanol is recovered from biomass and stage (ii) isrepeated up to four times.